Method for making a light energized tissue adhesive

ABSTRACT

Consistent with the present invention, tissue adhesive compositions and an associated laser exposure system are provided for bonding or sealing biological tissues. The compositions are comprised of chemically derivatized soluble collagen which is formulated to concentrations ranging from 300 mg/ml (30%) to 800 mg/ml (80%) collagen protein. In particular, Type I collagen, for example, is first prepared by extraction from bovine or porcine hide and purified. The collagen preparations are then chemically derivatized with sulfhydryl reagents to improve cohesive strength and with secondary derivatizing agents, such as carboxyl groups, to improve the adhesive strength of the solder to the tissue. The compositions are then formed into viscous solutions, gels or solid films, which when exposed to energy generated from an infrared laser, for example, undergo thermally induced phase transitions. Solid or semi-solid protein compositions become less viscous enabling the high concentration protein to penetrate the interstices of treated biological tissue or to fill voids in tissue. As thermal energy is released into the surrounding environment, the protein compositions again become solid or semi-solid, adhering to the treated tissue or tissue space.

BACKGROUND OF THE INVENTION

Tissue joining and sealing utilizing sutures and staples is the keystonein modern surgical procedures. An alternative approach utilizes a laserto expose a glue or solder to thereby join adjacent tissues. In manyrespects, such laser assisted tissue soldering is superior toconventional suture or staple-based methods. The advantages are speed,reduced infection, acceleration in healing, better cosmetic appearanceand ease of use particularly in laparoscopic surgery where watertightness and limited access or small size of the repair are important.Laser assisted tissue welding often requires glues or solders forpromoting strong bonds, forming a bridge between two apposed tissuesections. The ideal solder is chemically and mechanically matched to thetissue. As a result the solder is strong but stretches and grows withthe tissue because of good modulus match. Chromophores mixed with thesolder can assist in confining the deposited energy and reduce theimpact of tissue imperfections. These additives, however, may be toxicand cause pernicious effects.

The need to provide reliable sutureless closures is paramount toreducing morbidity and mortality rates and lowering health care costs.Achieving complete anastomotic integrity without damaging native tissueswill provide the general surgeon with a very powerful tool. For example,laser assisted tissue welding can be used in heart surgery to repaircongenital defects thereby eliminating blood loss, the main cause ofmortality in these procedures. Tissue welding can also benefit patientswith coagulation abnormalities, those suffering from dissection of theaorta and those undergoing minimally invasive coronary artery by-passgrafting (CABG). Laser assisted anastomoses will prevent post-operativeleakage in bowel and esophageal repair. It will provide a means forrepairing damage to articular cartilage, a common problem affectingjoints of millions of people. Sutureless transplantation ofosteochondral autografts can reduce pain and recovery time andeventually eliminate the need for total joint replacement. The use of asolder will further reduce dead spaces between circular grafts andprovide a bridge for optimizing the different mechanical properties ofdonor to recipient hyaline cartilage. Laser assisted tissue welding canalso be used to repair meniscal and treat osteoarthritis and spinal discinjuries and are among other applications of this invention.

Methods reported to date have not gained clinical acceptance. The majorreasons include the high level of surgeon skill that is required, thestrength, toxicity and resorbability of the tissue solder, the potentialfor irreparable laser damage and cost of the laser system.

SUMMARY OF THE INVENTION

Consistent with the present invention, a suitable solder and associatedlaser welding system are provided which avoid the shortcomings ofconventional systems described above. For example, the biologicalsolders and sealants consistent with the present invention arebiodegradable and do not require chormophores or dyes to promoteadhesion. Further, consistent with the present invention, the lasersystem provides accurate temperature control to eliminate peripheraltissue damage, damage to stay sutures (if required), and large areaexposure to reduce treatment time. Further, the use of a feedbackcontroller reduces required surgeon skill. The laser system can becomprised of inexpensive off-the-shelf components and has been designedto be compact, nonintrusive in a surgical setting, inexpensive tomanufacture and user friendly.

It is believed that the laser energy disrupts the three-dimensionalstructure of collagen fibers found in tissues, promoting tissuecrosslinking and improving cohesive strength. The films or gels are easyto apply and fix to the tissue surface.

Further consistent with the present invention, a method is provided forpreparing suitable protein compositions for use with the laser system.The compositions are comprised of chemically derivatized solublecollagen, which is formulated to concentrations ranging from 300 mg/ml(30%) to 800 mg/ml (80%) collagen protein. In particular, Type Icollagen, for example, is first prepared by extraction from animal hide,skin or connective tissue and purified. The collagen preparations arethen chemically derivatized with sulfhydryl reagents to improve cohesivestrength and with secondary derivatizing agents, such as carboxylgroups, to improve the adhesive strength of the solder to the tissue.The compositions are then formed into liquid, gels or solid films whichwhen exposed to energy generated from an infrared laser, for example,undergo thermally induced phase transitions. Solid or semi-solid proteincompositions become less viscous enabling the high concentration proteinto penetrate the interstices of treated biological tissue or to fillvoids in tissue. As thermal energy is released into the surroundingenvironment, the protein compositions again become solid or semi-solid,adhering to the treated tissue or tissue space.

In accordance with an additional feature of the present invention,minute quantities of any derivative of commercially available, medicalgrade cyanoacrylate can be applied to fix and appose tissue edges. Nexta layer of the high concentration collagen can be applied adjacent toand on the surface of the tissue to be bonded. This top layer istypically exposed to a infrared laser to melt, denature, and mix the twocomponents to promote both chemical and mechanical bonds to the tissueas well as enhancing intrinsic strength of the composite solder.Infrared laser exposure increases temperature at the bonding site.Feedback control of the laser can be used to adjust solder temperaturefor optimizing the bonding mechanism to the tissue while preventingthermal damage to adjacent healthy tissue.

It is believed that high concentration collagen compositions describedin this invention have an increased volume of linkages to improve thestrength performance of the solder while modulating the thermal melttemperatures and solder viscosities. Laser exposure parameters werechosen to rapidly flow the solder and promote physical contact of thesolder with the tissue. These parameters include choice of an operatingwavelength in the infrared range, power density, pulsed or cw mode,exposure times and tissue temperature. The following examples areillustrative of the invention but are not intended to limit the scopethereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of the present invention will be apparent from the followingdetailed description of the presently preferred embodiments thereof,which description should be considered in conjunction with theaccompanying drawings in which:

FIG. 1 illustrates an application of a tissue solder, which can be inliquid, gel or solid form, consistent with the present invention to bondtwo adjacent tissues;

FIG. 2 illustrates an additional application of a tissue solder, whichcan be in liquid, gel or solid form consistent with the presentinvention to bond two adjacent tissues;

FIG. 3 illustrates an application of a tissue solder, which can be inliquid, gel or solid form consistent with the present invention to bondinner surfaces two adjacent tissues;

FIG. 4 illustrates an additional application of a tissue solder, whichcan be in liquid, gel or solid form consistent with the presentinvention to bond inner surfaces of two adjacent tissues;

FIG. 5 illustrates a temperature monitoring and laser control system inaccordance with a further feature of the present invention;

FIG. 6 illustrates an adhesive patch consistent with a further aspect ofthe present invention;

FIG. 7 illustrates a mold used to make the patches shown in FIGS. 6, 8and 9;

FIGS. 8 and 9 illustrates additional exemplary adhesive patchesconsistent with the present invention; and

FIG. 10 illustrates a graph of minimum strengths of welded tissues.

DETAILED DESCRIPTION OF INVENTION

The family of proteins known as collagens are ubiquitous in nature andpredominant components of all connective tissues. Type I collagen is themost abundant of the twenty characterized collagens and is commonly usedas a biomaterial for medical devices. Collagen-based devices includehemostats, bulking agents, eye shields, punctal plugs, adhesives, drugdelivery systems, and others. Collagen is well known to be abiocompatible and safe biomaterial.

Consistent with the present invention, collagen is isolated and purifiedfrom various tissue sources including dermis, tendon and ligaments.While collagen is currently prepared from human dermis, most commercialproducts are prepared using collagen from animal sources. Once a pureform of collagen is prepared, it can be treated in several ways toprovide a suitable base protein for use as an adhesive or sealant. Thesetreatments include chemical derivitazation to add functional or reactivegroups as well as reduce the pKa of the solder to a more acidic value,and gelatinizing the collagen to create a viscous liquid, gel or solidfilm having a relatively high protein concentration.

The exemplary solder formulations described herein are suitable forinfrared laser activation and include chemically derivatized bovine TypeI collagen formed into a liquid, gel or solid film. Collagen was chosendue to its long history as a safe, biocompatible biomaterial and due toits ability to be chemically functionalized into a base formulation withunique cohesive and adhesive characteristics.

1. Exemplary Preparation of Pure Type I Collagen Solutions

Pure, Type I collagen was prepared from calf corium. Corium was cut intosmall pieces approximately 1 cm², soaked in reagent alcohol andextensively washed with sterile, deionized water. The washed pieces werethen swollen in acetic acid and digested using porcine mucosal pepsin(Sigma Chemical Company, 1:60,000 crystallized and lyophilized).Following 1 or more pepsin treatments, residual pepsin was removed bydialysis and Type I collagen isolated by differential saltprecipitation. Purified Type I collagen was dialyzed against acetic acidand filtered through 0.45μ and 0.22μ filters. Solder formulationsprepared from calf collagen were not successful unless the solutionswere extensively dialyzed to remove residual salt (NaCl) and filteredthrough a 0.22μ filter. It is believed that 0.22μ filtration is requiredto produce monomolecular collagen for subsequent chemicalderivatization. Details of pure, soluble Type I bovine collagen aredescribed in U.S. Pat. Nos. 4,713,446; 5,104,957; 5,219,895; 5,476,515;5,631,243, each of which is incorporated by reference herein. Anexemplary collagen purification process is provided below.

a. Skin preparation

1) Remove about 25 grams of bovine skin (split calf hide) and cut intosmall pieces using a scalpel and blade. Wash the skin with sterile,deionized water.

2) Cut the skin into small pieces using a scalpel blade. Weigh piecesand place in a 4 liter beaker containing 3 liters of 0.5M acetic acid.(This is 28.6 ml of concentrated acetic acid per liter or a total ofabout 86 ml. Add the 86 ml of acetic acid to 3 liters of water).

3) Attach stirrer and stir for 18 hours.

4) Add 2% pepsin to the container. First weigh out 0.5 grams of pepsinand dissolve in 50 ml of deionized water. Then add to the beaker.

5) Continue stirring for 12 hrs. If all of the skin pieces have notdispersed into the acetic acid, add another aliquot of 1% pepsin or 0.25gram of pepsin powder. Stir for 12 hrs.

6) Filter the suspension through cheesecloth to remove pieces of skinthat have not dispersed into the acetic acid. (remove a small sample foranalysis-40 ml in a 50 ml centrifuge tube and place in refrigerator.

7) Add solid NaCl (salt) to 0.8 M. This is 140 grams. Add while stirringthe collagen solution. Stir for at least two hours to precipitatecollagen. Allow the precipitate to settle for 12 hrs.

8) Siphon-off the clear liquid leaving the collagen precipitate. Thencentrifuge the precipitate in centrifuge bottles and collect all theprecipitate. Weigh and redissolve in 3 liters of 0.5M acetic acid. (seeabove for details in preparing the acetic acid solution). Stir for 12hrs.

9) Take sample of solution, 40 ml in a 50 ml centrifuge tube and cool to5 C.

10) Again add solid NaCl to 0.8M. Same as above. Stir for at least 2 hrsor until the precipitate is dissolved. Allow precipitate to settle for12 hrs and centrifuge to collect the pellet. Weigh and redissolve in 0.1M acetic acid (add 12 ml of acetic acid to 2 liters of deionized water).

11) Stir for 12 hrs to redissolve the collagen. Then dialyze thecollagen solution using Spectrapore dialysis tubing, >30,000 molecularweight cut-off. This step will remove dissolved pepsin. Then dialyzeagainst 0.1M acetic acid.

12) Dialyzed volumes will then be filtered through 0.45μ and then 0.2μfilters and stored in 1 liter or 4 liter bottles in the refrigerator.This is the collagen stock solution for making collagen-based solderformulations.

2. Chemical derivatization of Pure Type I Collagen Solution

Derivatization was intended to provide functional groups to enhance bothcohesive and adhesive characteristics. For cohesive functionality, thiolgroups were attached by derivatization using 4-mercapto-1,8-naphthalicanhydride. Adjacent thiol groups react to form disulfide bonds betweencollagen molecules enhancing the cohesive properties of this solder.Immediately after derivatization with the 4-mercapto-1,8-naphthalicanhydride, monomolecular collagen is derivatized with glutaricanhydride. This reaction substitutes a COO⁻ for a NH₃ ⁺ making thecomposition anionic. It is believed that the net negative charge of thesolder will ionically interact with positive charged proteins intissues. This reduces the pKa of collagen to the acidic pH rangeresulting in a collagen preparation that remains soluble atphysiological pH. The derivatization is conducted such that theresultant solder will exhibit a thermal transition at approximately40-50° C. when exposed to IR laser energy.

To prepare derivatized collagen, adjust 2000 ml collagen solution at2.5-3.0 mg/ml to pH 8.5-9.5 using 10N and 1N NaOH while stirring at roomtemperature.

For derivatization to attach both SH⁻ and COO⁻ groups, 40 ml of analcohol saturated solution of mercapto naphthalic anhydride (6 mg/ml)was added to the collagen and mixed for 30 minutes at 2-8° C. and mixedfor 15 minutes. Solid glutaric anhydride at 10% collagen weight (600-800mg of glutaric anhydride) was added and mixed for another 30 minutes atroom temperature. The pH was held at pH 8.5-9.5 using 1N NaOH.

At the end of 30 minutes, the solution was centrifuged at 3500 rpm for10 minutes. The pH of the solution was adjusted to pH 6.8-7.8 asnecessary. This solution was lyophilized or air-dried.

Collagen was also prepared with glutaric anhydride alone or with noderivatization. In the latter case, acid soluble collagen wasprecipitated at neutral pH and lyophilized or air-dried.

3. Solder Formulations

Solder formulations were prepared from chemically derivatized Type Icollagen. Base compositions contained either COO⁻ functional groups orboth SH⁻ (thiol) and COO⁻ functional groups. The degree ofderivatization with SH⁻ functional groups was varied in attempts tomodulate cohesive characteristics. Remaining free amine groups on thenative collagen molecule were derivatized with COO⁻ groups. Thus,compositions contained approximately the same total net derivatizationwith SH⁻ and COO⁻ groups. The ratio of SH⁻/COO⁻ was intentionallyvaried. Base preparations contained only COO⁻ groups. Derivatizedcollagen solutions were then lyophilized (or air dried and powdered).Lyophilized derivatized collagens were formulated into viscouscompositions having from 30-80% collagen solids. Since collagentypically becomes saturated at less than 10% solids, novel techniqueswere developed to increase total collagen concentrations to 30-80%. Thiswas accomplished by mixing lyophilized or powdered collagen preparationsin 0.02M phosphate buffer at pH 6.8-7.8. Mixtures of approximately 50mg/ml were initially prepared and exposed to thermal energy, in thiscase microwave energy (output power of 1100 watts, 100% power for 3-5seconds). Microwave energy generates thermal energy causing thegelatinization of collagen. Other heating methods can be employed suchas direct application of a heat source at 50 C, for example. Lyophilizedor dried collagen was added to the gelatinized collagen solution andagain exposed to microwave energy. This sequence continued until thedesired collagen concentration was attained. The final viscous andadhesive formulation was centrifuged at 3500 rpm for 1 minute. The highconcentration preparations were then cast into films by pouring hot,centrifuged gelatinized collagen in molds approximately 1 mm thick, and4 cm square in size. Solidified gelatinized collagen films weresectioned into strips for use with the infrared laser system. Analternate method of preparing films was to gently roll the centrifugetubes used to deaerate the thick gelatinized collagen formulations. Thehot, thick formulation coated the inner surface of the centrifuge tubesin a uniform thickness. Films were removed form the tubes and sectionedinto strips for use with the infrared laser system. Increased SH⁻/COO⁻appeared to increase thermal transition temperature and cohesivecharacteristics. However, cohesive strength appeared to be only minimal.Attempts to enhance adhesiveness were made by decreasing the SH⁻/COO⁻ratio. This typically resulted in a reduced thermal transitiontemperature but with little improvement in adhesive characteristics. Itis postulated that a greater increase in anionicity may provide enhancedadhesiveness to tissues.

Additional solder formulations were prepared from collagen derivatizedwith glutaric anhydride only or from non-derivatized acid solublecollagen. In both cases, it was critical to adjust the gelatinizedcollagen pH to 6.8-7.8 prior to preparing solder films. Therefore,during the microwave gelatinization and prior to deaeration, the pH wasadjusted to 6.8-7.8 by addition of 10N and 1N NaOH. These collagen-basedsolders appeared to provide the best biomechanical and adhesivecharacteristics.

FIG. 1 illustrates an exemplary application of the solder or gel andassociated laser system consistent with the present invention. First andsecond tissues 110 and 120 to be joined together, are brought withinclose proximity with one another. A layer of gelatinized solder 130 tobe used as an adhesive is then provided overlying the location where thetissues are to be joined. The solder is then exposed to electromagneticradiation 140, which in this example, includes infrared light generatedby a laser. Upon activation by the laser light, the solder becomes lessviscous and therefore flows, for example, into region 150 at thejunction of the two tissues 110 and 120. The solder penetrates theinterstices of tissues 110 and 120 and fills voids located in region150. With further application of radiation, the derivatized collagenwithin the solder crosslinks as well as crosslinking to the underlyingtissues. Withdrawing the laser causes the solder to harden and becomesolid or semi-solid and adhering to the treated areas.

Direct bonding of many tissues can be achieved using the inventivederivatized collagen adhesive alone. Additional adhesive strength,however, can be obtained for bonding some vascular and peritonealtissues by providing a layer of cyanoacrylate containing material to thetissues prior to application of the derivatized collagen adhesive.

For example, as shown in FIG. 2, a layer of cyanoacrylate 210 (n-butylor n-octyl cyanoacrylates, for example) can be initially applied totissues 110 and 120. The cyanoacrylate layer is believed to interactwith NH₂ groups in protein molecules in tissues 110 and 120 to initiatenucleophiles, kicking-off monomer polymerization and bonding to proteinsurfaces. In particular, derivitized collagen formulations, as discussedabove, were combined with n-butyl or n-octyl cyanoacrylates, as shown inFIG. 2. The collagen-based solder, and the tissue surface, contain NH₂moieties to initiate polymerization leading to primary chemical bondingwith the polymerized cyanoacrylate. This event results in bonding of thesolder to the tissue surface. In addition, exposure of the collagensolder to the laser 140 produces enough heat to induce a phasetransition of the collagen-based solder from solid or gel to liquid tothereby permit more efficient mixing of the solder with thecyanoacrylate monomer and facilitate penetration of the collagen-basedsolder into the tissue surface. The former encourages monomer reactionwith both the solder and the tissue, and the latter results in somedegree of mechanical bonding of the solder with the interacting treatedtissue.

Alternative applications are next shown in FIGS. 3 and 4. In FIG. 3,derivatized solder layers 310 and 320 are applied to the inner surfacesof tissues 120 and 110, respectively, and exposed to laser light 140 atthe interface between tissues 110 and 120 to complete the weldingprocess. In FIG. 4, cyanoacrylate layers 410 and 420 are applied toinner surfaces of tissues 120 and 110, respectively. Next, derivatizedcollagen solder layers 430 and 440 are applied to layers 410 and 420,respectively, and exposed to laser light in a manner similar to thatdescribed above to thereby facilitate bonding of the two tissue layers.

FIG. 5 illustrates a laser control and monitoring apparatus 500 inaccordance with a further feature of the present invention. In apparatus500, an energy source 510, such as a semiconductor laser emitting lighthaving a wavelength in the infrared range, supplies electromagneticradiation to a delivery fiber 520. Preferably, the wavelength outputfrom energy source 510 is selected to correspond to an absorption peakof water to thereby effectuate a more efficient coupling of energy fromsource 510 to target 530 (including the above described solder disposedon a tissue to be joined or bonded together). A second fiber 540 havingan end in proximity to the target 530 directs electromagnetic radiationemitted by the target to a temperature sensor circuit 550. The sensedradiation is detected by circuit 550 which generates a sense signal inresponse to a temperature of the target. Control circuit 560 receivesthe sense signals and outputs a control signal 510 to thereby adjust anintensity of laser light output from source 510. Thus, the outputintensity of source 510 is controlled in accordance with the temperatureof target 530.

Examples of tissue welding applications consistent with the presentinvention will next be described.

EXAMPLE 1

Descending aorta sections were collected from juvenile pigs and storedfrozen. Prior to use the tissue, contained in a sealed bag, was thawed15-20 min in a water bath at room temperature (23° C.). The thawed aortawere cut with a standard single-edged razor blade into 1 cm×1 cmcoupons. Excess fascia and loose tissue were removed. The coupons weretransferred to a PBS 1×solution that was placed in an ice bath untiluse.

The coupons were cut into two 0.5 cm×1 cm pieces and assembled in a buttjoint configuration. The incision was first warmed with the laseroperating in the 1.4-1.5 micron wavelength range. Laser power densitywas approximately 10 W/cm2. A minute quantity of medical gradecyanoacrylate is applied sparingly to the apposed tissue edges and thickcollagen gels or films were then applied directly to the cut surfacesintended for the joint and subsequently exposed to the laser at acontrolled temperature of 55° C. The laser beam was scanned over theentire gel or film for a period of 2-3 min. The gels or films underwentthermal melting. The high concentration fluid covered the incision andpenetrated the tissue interstices. Rapid cooling was observed once thelaser beam was withdrawn. The melted protein/cyanoacrylate compositebecame solid and adherent to the tissue providing adequate sealing toprevent fluid leakage. To quantify the welded mechanical strength,tensile tests were performed on coupons of tissue as prepared by themethod described above. FIG. 10 summarizes the results. As shown on thefirst bar of the graph, the minimum strength of the welded tissue wascalculated using Laplace's law. An aorta subjected to 180 mmHg bloodpressure will achieve a wall stress of 1000 g/cm². From the experiment,it was found that the combining of the cyanoacrylate and collagen solderhad a strength 5 times the minimum required strength. However, thecollagen alone or the cyanoacrylate alone achieved a lower maximumstrength.

EXAMPLE 2

Several ⅜ diameter articular cartilage plugs were cut with a tubularharvester in the vicinity of bovine carpel joints. The defects werewarmed with the laser operating in the 1.4-1.5 micron wavelength range.Laser power density was approximately 10 W/cm2. Thick collagen-basedgels were immediately troweled into the defect by a spatula or dispensedwith a microsyringe. Care was taken to apply the collagen gel to allsurfaces of the implant prior to insertion into the defect. In mostcases the implant surfaces were exposed to the laser to liquefy thecollagen-based gel then immediately transferred to the defect. Thesurface temperature was controlled in the 55-60° C. range. After implantplacement additional gel was applied and melted with the laser attemperature controlled in the 55-60 ° C. range. Rapid cooling wasobserved once the laser beam was withdrawn and the solder formed a solidand adherent seal around the implant. Lift and torque tests wereperformed immediately following the laser treatment, after 10 min andafter overnight storage. Ninety five percent (95%) of the strength wasmeasured immediately following laser exposure with maximum strengthobserved within 2 hrs.

A collagen-based composition comprised of Type II collagen may bepreferred in the treatment of articular cartilage lesions. One method ofpreparing Type II collagen is described for example in U.S. Pat. No.5,840,848 incorporated by reference herein.

Although the above described have been discussed for vascular andcartilage type tissues, this invention is applicable for all tissues andwound closures. For other tissues one with ordinary skills would makeappropriate modifications to the above described laser operatingparameters.

Consistent with a further aspect of the present invention, a tissueadhesive patch 600 can be used to bond tissues instead of layers 130,for example, discussed above. A patch 600 consists of a mesh 610 formedof a polymer (e.g. nylon, polyester or polycarbonate), carbon or metalwire that is biologically compatible and non-irritating. As shown inFIG. 6, the mesh is comprised of a cojoinal network which isencapsulated in a derivatized collagen liquid, gel or solid 620, asdiscussed above. The mesh can be pre-treated with a combination ofetchants, acids or cleaning solutions to enhance the adhesiveness to thebase derivatized collagen 620, as discussed above. Patch 600 hasincreased strength, flexibility or desired electrical characteristicsdue to the presence of mesh 610. Alternatively, a plurality of fibers810 (made of biologically compatible and non-irritating material such asa polymer (e.g. nylon, polyester, polycarbonate), carbon or metal can bedispersed throughout layer 620, which can either be oriented randomly(FIG. 8) or coaligned (FIG. 9) to give additional strength, flexibility,uniform heating or electrical properties to the patch 600.

Patch 600 can be formulated by placing a structural element such asfibers 810 or mesh 610 in a recessed portion 710 of mold or template700, as shown in FIG. 7. Heated derivatized and concentrated collagen,as discussed above, is poured or otherwise supplied to recess 710 andallowed to cool. Patch 600 is then removed from opening 710 and can beexposed to laser light or other electromagnetic radiation to bond orweld tissues in a manner similar to that discussed above. By preciselyfilling opening 710, patch 600 can be formed with a uniform thicknessthereby assuring adequate and uniform bonding of the underlying tissueupon exposure.

Consistent with a further aspect of the present invention, collagenfibrils, fibers, or fiber bundles can be added to the base soldercomposition to encourage integration of cells and binding of cells fromhost tissue. Collagen fibrils may be prepared by reconstituting acidsoluble or acid soluble-pepsin digested collagen from human or non-humananimal sources. Collagen fibrils may be composed predominantly of Type Icollagen, Type I collagen/Type III collagen mixtures, Type II collagen,or other collagen Type mixtures. Acid soluble and acid soluble-pepsindigested collagen undergo spontaneous reconstitution (fibrillogenesis)when brought to neutral pH conditions. This can be accomplished bymixing the acid soluble or acid soluble-pepsin digested collagen withappropriate buffer solutions at room temperature or slightly elevatedtemperatures (up to 40 C) or dialysis against buffer solutions at roomtemperature or slightly elevated temperatures (up to 40 C) to bring thepH of the acid soluble or acid soluble-pepsin digested collagen toneutral pH. There are many literature publications describing optimalmethods for collagen fibril reconstitution. Another method of preparingintact collagen fibrils is described in U.S. Pat. Nos. 4,969,912 and5,332,802. Such dispersions, predominantly composed of intact collagenfibrils, may be derived from any intact connective tissue includingdermis, tendon, ligament, cartilage, or other such collagen containingconnective tissue. Any other method of preparing collagen fibrils,collagen fibers, or collagen fiber bundles may also be appropriate.

Collagen fibrils, fibers, or fiber bundles are added to the base soldercomposition following centrifugation to remove incorporated air(deaeration). The dispersed fibrils, fibers, or fiber bundles are gentlymixed with an appropriate mixing tool, i.e., spatula, mixing rod, etc.,into the viscous composition avoiding production of air bubbles. If airbubbles are again incorporated, the mixture may be re-centrifuged atlower speed. Higher speeds will result in settling of the fibrils,fiber, or fiber bundles necessitating remixing to form a homogeneouscomposition. Mixing of fibrils, fibers, or fibril bundles is performedwhile the high concentration solder is still flowable but at atemperature lower than 65 C and preferably lower than 50 C. Fibril,fiber, or fiber bundle supplemented solder is then cast into films ormembranes as described above. Alternately, the composition may be placedin an appropriate delivery system, i.e., syringe or tubular device, suchthat the solder is able to be delivered by exerting pressure to forcingit through a delivery orifice (caulking gun approach) in a continuousstream.

Collagen fibrils, fibers, or fiber bundles are added to the base soldercomposition at concentrations (wet weight/volume) ranging from 1%-30%,preferably 5%-20%, and more preferably, 7.5%-15%. Since the addition ofintact collagen fibrils, fibers, or fiber bundles increases the totalconcentration of the solder, it may be necessary to reduce theconcentration of the base solder.

It is expected that such fibril, fiber, or fiber bundle soldercompositions will encourage host cell integration, cell binding, andcell proliferation.

The invention being thus disclosed and described in connection with theillustrated embodiments, variations and modifications thereof will occurto those skilled in the art, and are intended to be included within thescope of the invention.

What is claimed is:
 1. A method of making a tissue adhesive, comprisingthe steps of: heating a solution including collagen derivatized with aCOO⁻ functional group to thereby gelatinize said derivatized collagen;adding derivatized collagen to said solution, said derivatized collagenbeing derivatized with a COO⁻ functional group; and repeating saidheating and adding steps until the derivatized collagen concentration insaid solution is from 300 mg/ml (30%) up to 800 mg/ml (80%), said addedderivatized collagen in said solution being gelatinized after saidrepeated heating steps.
 2. A method in accordance with claim 1, furthercomprising the step of extracting said collagen from a tissue sourceprior to said derivatizing step.
 3. A method in accordance with claim 2,wherein said tissue source includes an animal tissue.
 4. A method inaccordance with claim 1, wherein said collagen is further derivatizedwith 4-mercapto-1,8-naphthalic anhydride.
 5. A method in accordance withclaim 1, wherein said collagen is derivatized with glutaric anhydride.6. A method in accordance with claim 1, wherein said collagen isderivatized with an SH-functional group.
 7. A method in accordance withclaim 1, further comprising a step of adding a pH altering material tosaid to thereby adjust a pH of said solution to be within a range of6.8-7.8.
 8. A method in accordance with claim 1, further comprising thestep of deaerating said solution.
 9. A method in accordance with claim7, wherein said pH altering material includes NaOH.
 10. A method inaccordance with claim 8, further comprising the step of: adding amaterial to said solution, said material being selected from the groupof collagen fibrils, collagen fibers and collagen fiber bundles.
 11. Amethod in accordance with claim 1, further comprising the step ofsolidifying said solution.
 12. A method in accordance with claim 11,further comprising the step of sectioning said solidified solution intostrips.
 13. A method in accordance with claim 1, wherein said addedcollagen is dried collagen.
 14. A method in accordance with claim 1,wherein said added collagen is lyophilized.